The present invention relates to peptides for the treatment of neurodegenerative disorders and more specifically for the treatment of Huntington's disease (HD).
Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disease. The average age of onset is at 30-50 years of age. It is characterized by the progressive deterioration of cognitive and motor functions, with a fatal outcome after approximately 10-15 years of onset. HD prevalence varies between 0.5 per 100,000 in Japan, to 5-15 per 100,000 in USA and Europe. The disease was first described in 1872 by Dr. George Huntington, who published the first description of three characteristics of the disease: The heredity, the “tendency to insanity”, as he observed, and age related onset.
The mutant protein involved in HD, was discovered in 1993 and was named Huntingtin (Htt). Htt is a large (340-350 kD, 3144 aa) protein that was shown to be involved in a number of cellular functions such as transcription, gastrulation, neurogenesis, neurotransmission, axonal transport, neural positioning, and apoptosis. In healthy individuals, Htt contains between 6 to 34 polyQ repeats in the N-terminus (FIG. 6). In the mutant form of Htt (mHtt), there is an extension of the polyQ repeats; a patient carrying over 40 glutamine repeats will certainly develop HD. The polyQ expansion results in the selective loss of GABAergic medium spiny striatal (MSN) neurons as well as glutamatergic cortical neurons that project into the striatum. The loss of the GABAergic MSN results in a lack of inhibitory signals from the striatum to the globus pallidus and the substantia nigra, and therefore induces excitatory signals to the neocortex. This disequilibrium is considered to be the cause for the involuntary movement symptom of the disease.
MHtt expression leads to the formation of PolyQ aggregates and inclusions, transcriptional dysregulation, excessive stimulation of glutamate receptors leading to excitotoxic neuronal damage, and the induction of apoptosis. The reduction of normal Htt activity may also contribute to disease manifestation, since normal Htt plays an important role in neuronal protection and survival and neurogenesis.
Htt was the first neuronal protein discovered to be the subject of a proteolytic cleavage by caspases. Caspase cleavage of Htt occurs at defined sites for caspase-3 at amino acids 513 and 552, caspase-2 at amino acid 552, and caspase-6 at amino acid 586 (FIG. 6). Additionally, there are two caspase-3 consensus sites at amino acids 530 and 589 that appear to be silent. Some other proteases, such as calpains, participate in Htt cleavage (FIG. 6).
Although the cleavage occur both in normal and mutant Htt, the mutant form is more susceptible to proteolysis and generates N-terminus fragments that aggregate in the cytoplasm and nucleus of neuronal cells, preceding neurodegeneration. These toxic to fragments cause additional activation of caspase-6, thus creating a positive feedback cycle of caspase activation, and induction of apoptosis.
The significance of mHtt proteolysis in HD pathogenesis is manifested by the fact that inhibiting caspase cleavage of Htt reduces toxicity and aggregates formation.
In order to reduce mHtt proteolysis, synthetic caspase inhibitors were tested on different experimental models, and indeed showed the reduction of mHtt toxicity induced in neuronal cultures and in HD animal models [Kim, M. et al., The Journal of Neuroscience, 1999, 19, 964-973: Ona, V. O et al. Nature, 1999, 399, 263-267]. These synthetic inhibitors are pseudo substrates for active caspases. They are based on small, usually 3-4 aa long peptides, conjugated to carboxyterminal groups such as chloromethyl ketone (cmk), fluoromethyl ketone (fmk), or aldehyde (cho). These groups enable them to act as competitive inhibitors, increase the affinity to caspases and improve cell permeability and stability. These cell permeable inhibitors alkylate the active site cysteine of caspases and irreversibly block apoptosis by preventing caspase activation, substrate cleavage, and DNA ladder formation. However, most synthetic caspase inhibitors are hydrophobic and not very permeable, and could cause nonspecific toxic effects when added at concentrations required to inhibit intracellular caspases [Ona, V. O et al. Nature, 1999, 399, 263-267: Frydrych, I.; Toxicology in Vitro, Proceedings of the Scandinavian Society of Cell Toxicology 2007 Workshop, 2008, 22, 1356-1360; Zhu, S.; et al., Cell Death and Dis, Macmillan Publishers Limited, 2011, 2, e115; Chauvier, D. Cell Death Differ, 2006, 14, 387-391]. Experiments in knockout mice indicated that caspase-6 deficiency is not fatal or causes severe toxic effects.
Garcio et al (The Journal of Biological Chemistry, 273, (4): 371-376) teaches inhibition of human caspases by peptide based and macromolecular inhibitors. Nyormoi O et al., (Apoptosis. 2003 August; 8(4):371-6) teaches a synthetic peptide inhibitor of caspase 6.